Figure 1 - An image showing the three agar plates containing ampicillin and the transformed bacterial specimen NEB pGEM-gbr22 (left), saliva with no ampicillin (middle), and ampicilllin with the non-recombinant bacterial species NEB (right). None of the plates showed any growth.
Figure 2 - An image showing the agar plate with containing ampicillin and the transformed bacterial specimen NEB pGEM-gbr22 (left in Figure 1) nine hours later with a small colony of purple colored bacteria near the center.
Figure 3 - An image showing the two cultures Mohammed and I grew from the transformed bacteria in order to create a large enough amount of the cells (and therefore protein) to isolate from the solution and later purify.
Figure 4 - An image showing the two wet cell pellets isolated from the two flasks in Figure 3. The pellets were subsequently suspended in a 1X PBS working solution and lysozyme was added to break up the cell walls and release the protein from inside the bacterial cell membranes in order for further purification at a later date.
Lab Protein Purification
Figure 5 - An image of the two elutions Mohammed and I performed to conclude the purification of our protein.
Lab Protein Characterization
Figure 6 - An image of the gel used to preform the gel electrophoresis of the six samples gathered throughout the first two protein labs before drying. The molecular weight standard is on the very left.
Figure 6 - An image of the gel used to preform the gel electrophoresis of the six samples gathered throughout the first two protein labs after drying. The molecular weight standard is on the very left.
Protein Labs Report
Introduction
As scientists strive to study disease and the proteins associated with them, they must utilize efficient and low cost means by which they can produce their targeted proteins, a juxtaposition of necessities for researchers for almost all of history. However recent endeavors by multiple groups have created a process for protein replication and purification that has opened up protein study to more researchers than ever before.
By capitalizing bacteria’s extremely adaptive cellular machinery, researchers can utilize lab strains of E. coli, completely harmless to humans with their default DNA, as a source of cheap and self-replicating factories of foreign protein. This ability theoretically applies to proteins of any species. E. coli naturally expresses DNA in the form of plasmids, double stranded circular helixes of DNA residing in the cytoplasm, and can even eventually incorporate the genes of the plasmid into its genome. As long as the genes have the necessary binding sites to trigger transcription, E. coli will transcribe and produce the protein indiscriminately. Even eukaryotic genes, once converted to cDNA from a mature strand of mRNA, can reside in a plasmid. Through the use of ligands and heat shocking, researchers can induce E. coli to open pores in its cell membrane and accept foreign plasmids. Researchers can even designate the mechanism that triggers transcription of the protein and control whether or not the bacteria expresses the protein.(1,2)
Through the use of plasmids incorporating both the foreign protein and specific antibiotic resistance, researchers can treat their cultures with the antibiotic that the plasmid contains resistance for and ensure that the surviving cultures consist of only bacteria that have accepted the plasmid and therefore express the protein of interest. Once researchers produce a viable culture, that culture has the ability to replicate at an astounding rate and produce a huge amount of the desired protein.(2)
Once the culture has grown to a desirable degree, the researcher can collect a large volume of the cells and destroy the membranes (for a soluble protein) and prepare the cell products for purification. Since each of the molecules that make up a cell have unique a densities and solubility, a researcher can separate the desired protein from the non-soluble parts of the cell by centrifuging. By using a modified version of the desired protein with a tail of added histidines, the soluble portions consisting mostly of dissolved proteins can be treated with a resin-buffer mixture bound with a special conformation containing nickel. The tail of histidines attached to the desired protein will bind to the nickel. A purification column can then act as a matrix with which the resin can rest on and solutions of imidazole poured down the column will filter out proteins attached to the resin trapped by the matrix with a lower affinity than the desired protein. Eventually a solution with a relatively large amount of imidazole will release the desired protein from the resin which a research can collect.(1)
In order to ensure the purity of the sample, the collected solution of protein must be tested for contamination. By introducing the protein solution with a chemical such as sodium dodecyl sulfate, the proteins of the solution denature and acquire a charge. The sample of the protein can then undergo gel electrophoresis, a procedure which has proteins travel through a permeable gel of consistent density propelled by the influence of an electrical charge. The larger the sequence of the protein, the more overall negative a charge it possesses, and the further down the gel the protein will travel. By using a molecular weight sample, a collection of proteins with a known molecular weight, the researcher can identify if the target protein exists in the solution and whether or not the sample contains other proteins that were not removed by the purification.(1)
Methods
After acquiring a culture of a lab strain E. coli species, we split the bacteria into two collections: cells designated for transformation and cells designated for a control culture. We then introduced a plasmid containing the gbr22 protein and a gene encoding for ampicillin resistance, as well as the proper transcription factors, into the collection of the bacteria designated for transformation. These cells underwent exposure to SOC media in order to produce pores in the cells’ membranes through which the plasmid could enter the cytoplasm. We then proceeded to submerge the cells in ice and then heat shocked the cells in a 42˚C water bath for 45 seconds in order to increase the chance of the pores opening to accept the plasmids. Two agar plates were prepared with ampicillin; one was labeled “control” and the other “DNA.” The cells designated as “control” were placed on the agar plate of the same name while the cells that underwent transformation were placed on the plate labeled “DNA.” We incubated both plates in a 37˚C incubator overnight.
The next day, in order to create a large starter culture, we transferred cells that survived on the ampicillin treated plate labeled “DNA,” indicating that the cells expressed the plasmid introduced, into two flasks containing LB media and ampicillin. These flasks were allowed to incubate for 16-24 hours in a shaking incubator at 37˚C and 200-350 rpm. Once the cultures turned purple, indicating a large amount of bacteria expressing the plasmid, we poured the contents of the two flasks into two conical tubes and centrifuged the contents for 10 minutes at 5,000 rpm and 4˚C in order to separate the cells from the LB media and ampicillin solution. The solution was then discarded and the cells were re-suspended in 1X PBS buffer and transferred into micro centrifuge tubes. We added lysozyme into the cells in order to release the soluble protein from inside the cell.
We then centrifuged the cells in order to separate the soluble proteins from the non-soluble and heavier molecules of the cell such as DNA, hydrophobic proteins, and cell membrane lipids. We then collected the soluble liquid portion and discarded the rest. We then syringe filtered the solution in order to filter out the larger molecular matter left over from the cells. We then added Ni-NTA resin-buffer into the remaining solution and poured it into a Bio-Rad Econo chromatography column (Bio-Rad Laboratories; Hercules, CA). Using a wash solution with a low concentration of imidazole, we filtered out proteins through the matrix of the column that were not strongly bound to the nickel of the resin (the desired protein was due to a tail of histidines added to its sequence). We then filtered out the desired protein using an elution solution containing a relatively high concentration of imidazole. Elution 1 was then analyzed with a NanoDrop spectrophotometer (NanoDrop Products; Wilmington, DE) at the wavelength 280 nm and at the maximum wavelength of absorbance for the protein gbr22, 574 nm. Using Beer's Law and assuming the protein was the only one existing in solution. we calculated two estimates for the concentration of the protein in solution.
This solution, as well as five other samples taken from various steps of the purification process, was mixed with a loading buffer containing sodium dodecyl sulfate. The sodium dodecyl sulfate attached to the residues of the proteins and denatured them as well as adding a negative charge. We then filled a Mini-PROTEAN electrophoresis tank (Bio-Rad Laboratories; Hercules, CA) containing a precast polyacrylamide gel cassette with TGS 1X buffer. All six samples and a molecular weight standard were loaded into the wells of the precast polyacrylamide gel. The gel electrophoresis was allowed to run for 25 minutes at 200 volts. We then collected the gel and washed it 3 times for five minutes as well as applying imperial protein stain and letting it sit for an hour on the orbital shaker. We then drained the imperial stain and let the gel soak in clean water with a paper towel on top of it overnight on the orbital shaker in order to remove the excess stain. The next day we dried the gel in a heated vacuumed presser for an hour and a half at 75˚C on a gradient cycle.
Results
Figure 1 - An image showing the three agar plates containing ampicillin and the transformed bacterial specimen NEB pGEM-gbr22 (left), saliva with no ampicillin (middle), and ampicilllin with the non-recombinant bacterial species NEB (right). None of the plates showed any growth.
Figure 2 - An image showing the agar plate with containing ampicillin and the transformed bacterial specimen NEB pGEM-gbr22 (left in Figure 1) nine hours later with a small colony of purple colored bacteria near the center.
For an unknown reason, the transformed bacteria took twice as long as expected to express a visible amount of the purple protein gbr22. However a single colony did appear and continued to cultivate for the duration of the protein expression portion of the lab.
Figure 3 - An image showing the two cultures Mohammed and I grew from the transformed bacteria in order to create a large enough amount of the cells (and therefore protein) to isolate from the solution and later purify.
Both flasks acquired a purple color in the time expected. This purple color ensured us that a large number of the bacteria expressing gbr22 did indeed grow from the small samples placed into the flasks before incubation.
Figure 4 - An image showing the two wet cell pellets isolated from the two flasks in Figure 3. The pellets were subsequently suspended in a 1X PBS working solution and lysozyme was added to break up the cell walls and release the protein from inside the bacterial cell membranes in order for further purification at a later date.
Centrifuging removed the majority of the LB media/amp solution, leaving a solid aggregate of the transformed cells on the bottom. The cell pellets weighed 1.06 g (left) and .87 (right). The starter cultures contained exponentially more cells than the colonies used to create them.
Figure 5 - An image of the two elutions Mohammed and I performed to conclude the purification of our protein.
Elution 1 was considerably more purple than elution two. The protein detached from the resin by the first elution greatly exceeds that of the second.
Figure 6 - A snapshot of the first run of elution 1 in the NanoDrop spectrophotometer measuring absorbance of the protein solution at a wavelength of 280 nm.
Figure 7 - A snapshot of the second run of elution 1 in the NanoDrop spectrophotometer measuring absorbance of the protein solution at a wavelength of 280 nm.
Figure 8 - A snapshot of the first run of elution 1 in the NanoDrop spectrophotometer measuring absorbance of the protein solution at the maximum wavelength of absorbance for the protein gbr22, 574 nm.
Figure 9 - A snapshot of the second run of elution 1 in the NanoDrop spectrophotometer measuring absorbance of the protein solution at the maximum wavelength of absorbance for the protein gbr22, 574 nm.
Using the information gathered from the spectrophotometer and assuming the solution contained only the protein gbr22, we calculated two estimates for the concentration of the protein in the solution, one from each wavelength used.
Molecular weight of gbr22 = 25,794.2 g
Beer’s Law:
c= A/εb
Where:
A is absorbance
ε is molar absorptivity (with units L mol-1 cm-1)
b is path length (with unit cm)
c is concentration (with units mol/L)
Avg. absorbance at the wavelength 280 nm = .0595 ; ε = 38850 ; b = .100 cm
(.0594)/(38850 X 0.100 cm) = 1.53X10^-5 M
(1.53X10^-5 mol)/(1.00 L) X (1.00 L)/(1.00X10^3 ml) X (5.00 ml) X (25,794.2 g)/(1.00 mol) = .00197 g
Avg. absorbance at the maximum absorbance wavelength 574 nm = .0740 ; ε = 118300 ; b = 0.100 cm
(.0740)/(118300 X 0.100 cm) = 6.26X10^-6 M
(6.26X10^-6 mol)/(1.00 L) X (1.00 L)/(1.00X10^3 ml) X (5.00 ml) X (25,794.2 g)/(1.00 mol) = .000807 g
Figure 10 - An image of the gel used to preform the gel electrophoresis of the six samples gathered throughout the first two protein labs before drying. The molecular weight standard is on the very left. (From left to right: the molecular weight standard, samples 1-6.)
Figure 11 - An image of the gel used to preform the gel electrophoresis of the six samples gathered throughout the first two protein labs after drying. The molecular weight standard is on the very left. (From left to right: the molecular weight standard, samples 1-6.)
Solutions in each well from left to right:
Well 1 --> Empty
Well 2 --> Molecular Weight Standard
Well 3 --> Sample 1
Well 4 --> Sample 2
Well 5 --> Sample 3
Well 6 --> Sample 4
Well 7 --> Sample 5
Well 8 --> Sample 6
Wells 9-10 --> Empty
Figure 12 - A template of how the molecular weight standard should look for the the type of gel we used. It includes the kDa (or kiloDaltons) of the proteins in each band.
The purity for this protein purification stood at about 30%. Using the molecular weight standard we can assume the protein has a molecular weight around 25,000 g.
Discussion
On the surface every procedure, with the exception of the transformed bacteria taking slightly longer than expected to cultivate, seemed to go exactly as expected. However the gel electrophoresis of the protein showed contamination. Most likely this contamination occurred because of an inadequate wash of the chromatography column before gathering the supposed purified protein. The bacteria may have also expressed proteins with a naturally high affinity for the Ni-NTA resin-buffer used. Since the second elution showed no visible signs of the purple protein gbr22, we can assume the mass majority of the protein did indeed release with the first elution. Since our purity stood at about 30%, we can expect the yield of the gbr22 protein at around 1/3 of the yield estimated by the NanoDrop spectrophotometer.
Conclusion
We were able to utilize the ability of E. coli to transcribe proteins not native to their genome through the introduction of recombinant plasmids in order to produce a large quantity of our desired protein, gbr22. We then lysed the cells to release the protein from the cytosol and bound the protein to the Ni-NTA resin buffer. We then purified the protein in a chromatography column and analyzed the solution with a NanoDrop spectrophotometer. Lastly we put the protein through a gel electrophoresis which revealed that the protein solution was contaminated and the protein purification failed. The next step would be to refine the techniques used for protein purification until they prove consistently successful. Then we can produce large amounts of a target protein pertaining to a certain illness or pathogen. These proteins can then be purified for evaluation in ligand-protein assays consisting of ligands virtual screened for affinity with the target protein.
References
1. Protein Expression and Purification Core Facility. http://www.embl.de/pepcore/pepcore_services/index.html (accessed April 14). 2. Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods2008,5 (2), 135-46.
Lab Protein Expression
Lab Protein Purification
Lab Protein Characterization
Protein Labs Report
Introduction
As scientists strive to study disease and the proteins associated with them, they must utilize efficient and low cost means by which they can produce their targeted proteins, a juxtaposition of necessities for researchers for almost all of history. However recent endeavors by multiple groups have created a process for protein replication and purification that has opened up protein study to more researchers than ever before.
By capitalizing bacteria’s extremely adaptive cellular machinery, researchers can utilize lab strains of E. coli, completely harmless to humans with their default DNA, as a source of cheap and self-replicating factories of foreign protein. This ability theoretically applies to proteins of any species. E. coli naturally expresses DNA in the form of plasmids, double stranded circular helixes of DNA residing in the cytoplasm, and can even eventually incorporate the genes of the plasmid into its genome. As long as the genes have the necessary binding sites to trigger transcription, E. coli will transcribe and produce the protein indiscriminately. Even eukaryotic genes, once converted to cDNA from a mature strand of mRNA, can reside in a plasmid. Through the use of ligands and heat shocking, researchers can induce E. coli to open pores in its cell membrane and accept foreign plasmids. Researchers can even designate the mechanism that triggers transcription of the protein and control whether or not the bacteria expresses the protein.(1,2)
Through the use of plasmids incorporating both the foreign protein and specific antibiotic resistance, researchers can treat their cultures with the antibiotic that the plasmid contains resistance for and ensure that the surviving cultures consist of only bacteria that have accepted the plasmid and therefore express the protein of interest. Once researchers produce a viable culture, that culture has the ability to replicate at an astounding rate and produce a huge amount of the desired protein.(2)
Once the culture has grown to a desirable degree, the researcher can collect a large volume of the cells and destroy the membranes (for a soluble protein) and prepare the cell products for purification. Since each of the molecules that make up a cell have unique a densities and solubility, a researcher can separate the desired protein from the non-soluble parts of the cell by centrifuging. By using a modified version of the desired protein with a tail of added histidines, the soluble portions consisting mostly of dissolved proteins can be treated with a resin-buffer mixture bound with a special conformation containing nickel. The tail of histidines attached to the desired protein will bind to the nickel. A purification column can then act as a matrix with which the resin can rest on and solutions of imidazole poured down the column will filter out proteins attached to the resin trapped by the matrix with a lower affinity than the desired protein. Eventually a solution with a relatively large amount of imidazole will release the desired protein from the resin which a research can collect.(1)
In order to ensure the purity of the sample, the collected solution of protein must be tested for contamination. By introducing the protein solution with a chemical such as sodium dodecyl sulfate, the proteins of the solution denature and acquire a charge. The sample of the protein can then undergo gel electrophoresis, a procedure which has proteins travel through a permeable gel of consistent density propelled by the influence of an electrical charge. The larger the sequence of the protein, the more overall negative a charge it possesses, and the further down the gel the protein will travel. By using a molecular weight sample, a collection of proteins with a known molecular weight, the researcher can identify if the target protein exists in the solution and whether or not the sample contains other proteins that were not removed by the purification.(1)
Methods
After acquiring a culture of a lab strain E. coli species, we split the bacteria into two collections: cells designated for transformation and cells designated for a control culture. We then introduced a plasmid containing the gbr22 protein and a gene encoding for ampicillin resistance, as well as the proper transcription factors, into the collection of the bacteria designated for transformation. These cells underwent exposure to SOC media in order to produce pores in the cells’ membranes through which the plasmid could enter the cytoplasm. We then proceeded to submerge the cells in ice and then heat shocked the cells in a 42˚C water bath for 45 seconds in order to increase the chance of the pores opening to accept the plasmids. Two agar plates were prepared with ampicillin; one was labeled “control” and the other “DNA.” The cells designated as “control” were placed on the agar plate of the same name while the cells that underwent transformation were placed on the plate labeled “DNA.” We incubated both plates in a 37˚C incubator overnight.
The next day, in order to create a large starter culture, we transferred cells that survived on the ampicillin treated plate labeled “DNA,” indicating that the cells expressed the plasmid introduced, into two flasks containing LB media and ampicillin. These flasks were allowed to incubate for 16-24 hours in a shaking incubator at 37˚C and 200-350 rpm. Once the cultures turned purple, indicating a large amount of bacteria expressing the plasmid, we poured the contents of the two flasks into two conical tubes and centrifuged the contents for 10 minutes at 5,000 rpm and 4˚C in order to separate the cells from the LB media and ampicillin solution. The solution was then discarded and the cells were re-suspended in 1X PBS buffer and transferred into micro centrifuge tubes. We added lysozyme into the cells in order to release the soluble protein from inside the cell.
We then centrifuged the cells in order to separate the soluble proteins from the non-soluble and heavier molecules of the cell such as DNA, hydrophobic proteins, and cell membrane lipids. We then collected the soluble liquid portion and discarded the rest. We then syringe filtered the solution in order to filter out the larger molecular matter left over from the cells. We then added Ni-NTA resin-buffer into the remaining solution and poured it into a Bio-Rad Econo chromatography column (Bio-Rad Laboratories; Hercules, CA). Using a wash solution with a low concentration of imidazole, we filtered out proteins through the matrix of the column that were not strongly bound to the nickel of the resin (the desired protein was due to a tail of histidines added to its sequence). We then filtered out the desired protein using an elution solution containing a relatively high concentration of imidazole. Elution 1 was then analyzed with a NanoDrop spectrophotometer (NanoDrop Products; Wilmington, DE) at the wavelength 280 nm and at the maximum wavelength of absorbance for the protein gbr22, 574 nm. Using Beer's Law and assuming the protein was the only one existing in solution. we calculated two estimates for the concentration of the protein in solution.
This solution, as well as five other samples taken from various steps of the purification process, was mixed with a loading buffer containing sodium dodecyl sulfate. The sodium dodecyl sulfate attached to the residues of the proteins and denatured them as well as adding a negative charge. We then filled a Mini-PROTEAN electrophoresis tank (Bio-Rad Laboratories; Hercules, CA) containing a precast polyacrylamide gel cassette with TGS 1X buffer. All six samples and a molecular weight standard were loaded into the wells of the precast polyacrylamide gel. The gel electrophoresis was allowed to run for 25 minutes at 200 volts. We then collected the gel and washed it 3 times for five minutes as well as applying imperial protein stain and letting it sit for an hour on the orbital shaker. We then drained the imperial stain and let the gel soak in clean water with a paper towel on top of it overnight on the orbital shaker in order to remove the excess stain. The next day we dried the gel in a heated vacuumed presser for an hour and a half at 75˚C on a gradient cycle.
Results
For an unknown reason, the transformed bacteria took twice as long as expected to express a visible amount of the purple protein gbr22. However a single colony did appear and continued to cultivate for the duration of the protein expression portion of the lab.
Both flasks acquired a purple color in the time expected. This purple color ensured us that a large number of the bacteria expressing gbr22 did indeed grow from the small samples placed into the flasks before incubation.
Centrifuging removed the majority of the LB media/amp solution, leaving a solid aggregate of the transformed cells on the bottom. The cell pellets weighed 1.06 g (left) and .87 (right). The starter cultures contained exponentially more cells than the colonies used to create them.
Elution 1 was considerably more purple than elution two. The protein detached from the resin by the first elution greatly exceeds that of the second.
Using the information gathered from the spectrophotometer and assuming the solution contained only the protein gbr22, we calculated two estimates for the concentration of the protein in the solution, one from each wavelength used.
Molecular weight of gbr22 = 25,794.2 g
Beer’s Law:
c= A/εb
Where:
A is absorbance
ε is molar absorptivity (with units L mol-1 cm-1)
b is path length (with unit cm)
c is concentration (with units mol/L)
Avg. absorbance at the wavelength 280 nm = .0595 ; ε = 38850 ; b = .100 cm
(.0594)/(38850 X 0.100 cm) = 1.53X10^-5 M
(1.53X10^-5 mol)/(1.00 L) X (1.00 L)/(1.00X10^3 ml) X (5.00 ml) X (25,794.2 g)/(1.00 mol) = .00197 g
Avg. absorbance at the maximum absorbance wavelength 574 nm = .0740 ; ε = 118300 ; b = 0.100 cm
(.0740)/(118300 X 0.100 cm) = 6.26X10^-6 M
(6.26X10^-6 mol)/(1.00 L) X (1.00 L)/(1.00X10^3 ml) X (5.00 ml) X (25,794.2 g)/(1.00 mol) = .000807 g
Solutions in each well from left to right:
Well 1 --> Empty
Well 2 --> Molecular Weight Standard
Well 3 --> Sample 1
Well 4 --> Sample 2
Well 5 --> Sample 3
Well 6 --> Sample 4
Well 7 --> Sample 5
Well 8 --> Sample 6
Wells 9-10 --> Empty
The purity for this protein purification stood at about 30%. Using the molecular weight standard we can assume the protein has a molecular weight around 25,000 g.
Discussion
On the surface every procedure, with the exception of the transformed bacteria taking slightly longer than expected to cultivate, seemed to go exactly as expected. However the gel electrophoresis of the protein showed contamination. Most likely this contamination occurred because of an inadequate wash of the chromatography column before gathering the supposed purified protein. The bacteria may have also expressed proteins with a naturally high affinity for the Ni-NTA resin-buffer used. Since the second elution showed no visible signs of the purple protein gbr22, we can assume the mass majority of the protein did indeed release with the first elution. Since our purity stood at about 30%, we can expect the yield of the gbr22 protein at around 1/3 of the yield estimated by the NanoDrop spectrophotometer.
Conclusion
We were able to utilize the ability of E. coli to transcribe proteins not native to their genome through the introduction of recombinant plasmids in order to produce a large quantity of our desired protein, gbr22. We then lysed the cells to release the protein from the cytosol and bound the protein to the Ni-NTA resin buffer. We then purified the protein in a chromatography column and analyzed the solution with a NanoDrop spectrophotometer. Lastly we put the protein through a gel electrophoresis which revealed that the protein solution was contaminated and the protein purification failed. The next step would be to refine the techniques used for protein purification until they prove consistently successful. Then we can produce large amounts of a target protein pertaining to a certain illness or pathogen. These proteins can then be purified for evaluation in ligand-protein assays consisting of ligands virtual screened for affinity with the target protein.
References
1. Protein Expression and Purification Core Facility. http://www.embl.de/pepcore/pepcore_services/index.html (accessed April 14).
2. Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods 2008, 5 (2), 135-46.